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Cell, Tumor, and Stem Cell Biology

Subcellular Localization of Sigma-2 Receptors in Breast Cancer Cells Using Two-Photon and Confocal Microscopy

Chenbo Zeng, Suwanna Vangveravong, Jinbin Xu, Katherine C. Chang, Richard S. Hotchkiss, Kenneth T. Wheeler, Duanwen Shen, Zhi-Ping Zhuang, Hank F. Kung and Robert H. Mach
DOI: 10.1158/0008-5472.CAN-06-3803 Published July 2007

Abstract

Sigma-2 receptor agonists have been shown to induce cell death via caspase-dependent and caspase-independent pathways. Unfortunately, there is little information regarding the molecular function of sigma-2 receptors that can explain these results. In this study, two fluorescent probes, SW107 and K05-138, were used to study the subcellular localization of sigma-2 receptors by two-photon and confocal microscopy. The results indicate that sigma-2 receptors colocalize with fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum, and the plasma membrane in both EMT-6 mouse and MDA-MB-435 human breast cancer cells. The fluorescent probe, K05-138, was internalized rapidly, reaching a plateau of fluorescent intensity at 5 min. The internalization of K05-138 was reduced ∼40% by phenylarsine oxide, an inhibitor of endocytosis. These data suggest that sigma-2 ligands are internalized, in part, by an endocytotic pathway. The localization of sigma-2 receptors in several organelles known to have a role in both caspase-dependent and caspase-independent pathways of cell death supports the conclusions of previous studies suggesting that sigma-2 receptor ligands should be evaluated as potential cancer chemotherapeutic agents. [Cancer Res 2007;67(14):6708–16]

  • sigma-2 receptor
  • subcellular localization
  • confocal microscopy
  • two-photon microscopy
  • breast cancer cell

Introduction

Cancer cells are often characterized by an overexpression of membrane-bound or intracellular receptors that can serve as a molecular target for diagnostic or treatment purposes. Examples of the former include somatostatin, bombesin, and Her-2/neu receptors, which serve as the molecular targets of octreotide, peptide-based bombesin antagonists, and herceptin for the treatment of neuroendocrine and breast cancer ( 1– 3). The most prominent example of the latter is the estrogen receptor, a nuclear receptor that is used to determine if breast cancer patients should be treated with the antiestrogen tamoxifen ( 4).

There is a significant amount of experimental evidence to suggest that sigma receptors are overexpressed in a variety of human and rodent tumors ( 5– 7) and likely play an important role in cancer biology ( 8). Sigma receptors are a class of proteins that were originally thought to be a subtype of the opiate receptors ( 9). Subsequent studies revealed that sigma binding sites represent a distinct class of receptors ( 10, 11). There are two well-characterized subtypes of sigma receptors, sigma-1 and sigma-2. The sigma-1 receptors have a molecular weight of ∼25 kDa, whereas the sigma-2 receptors have a molecular weight of ∼21.5 kDa. The sigma-1 receptor gene has been cloned from guinea pig liver, human placental choriocarcinoma, rat brain, and mouse kidney ( 12– 14). Ligands having a high affinity for sigma-1 receptors have shown promise as radiotracers for imaging melanoma ( 15).

A number of studies have reported that the sigma-2 receptor is a potential receptor-based biomarker of the proliferative status of solid tumors. For example, studies using a tissue culture model of mouse mammary adenocarcinoma cells have shown that sigma-2 receptors were expressed ∼10 times higher in proliferating (P) tumor cells than in the corresponding quiescent (Q) tumor cells ( 16). A subsequent study in solid tumor xenografts of the same tumor cell lines showed a positive correlation between the sigma-2 receptor density and the P:Q ratio measured by flow cytometry ( 17). The agreement between the solid tumor and tissue culture data indicates that the expression of sigma-2 receptors is likely a reliable biomarker of the proliferative status of solid tumors. Finally, radiolabeled sigma-2 selective ligands developed in our laboratory have shown promise in imaging studies of murine models of breast cancer, further suggesting that radioligands having a high affinity and high selectivity for sigma-2 receptors have the potential to noninvasively image the proliferative status of solid tumors in vivo with positron emission tomography (PET; refs. 18– 20).

Recent studies have also shown that sigma-2 receptor ligands induce apoptosis in the human breast tumor cell line, MCF-7 ( 21), human neuroblastoma cell line, SK-N-SH ( 22), and murine fibrosarcoma cell line, WEHI-S ( 23). Although the mechanism of cell death is largely unknown, several studies have revealed that sigma-2 receptor ligands induce apoptosis by caspase-dependent ( 22) and/or caspase-independent pathways ( 21, 23). In SK-N-SH neuroblastoma cells, sigma-2 ligands reduced mitochondrial membrane potential and induced caspase-dependent apoptosis, suggesting that sigma-2 receptors play a role in the intrinsic apoptotic pathway. Caspase-independent cell death may involve lysosome leakage, cathepsin activation, and oxidative stress ( 23). However, it is not known whether sigma-2 receptor ligands function by physically interacting directly with receptors residing in the mitochondria and lysosomes or via a downstream signaling mechanism. Information regarding the subcellular localization of sigma-2 receptors should provide valuable insight into the mechanisms and functions of sigma-2 receptors in cell death and proliferation.

In the present study, two new fluorescent ligands, SW107 and K05-138, having a high affinity for sigma-2 receptors, were used to examine the subcellular localization of sigma-2 receptors in both mouse EMT6 and human MDA-MB-435 breast cancer cells using two-photon and confocal microscopy. Coregistration studies with well-characterized fluorescent markers of organelles suggest that sigma-2 receptors are located in the mitochondria, lysosomes, endoplasmic reticulum, and plasma membrane. The localization of sigma-2 receptors in organelles known to play a key role in both caspase-dependent and caspase-independent pathways of cell death provides further support for the investigation of sigma-2 selective ligands as potential cancer chemotherapeutic agents.

Materials and Methods

Resources. MitoTracker Red CMXRos, ER-Tracker Red dye, LysoTracker Red DND-99, FM 1-43FX, and FM 4-64FX were purchased from Invitrogen Corporation. [3H]-pentazocine (31.6 Ci/mmol) was purchased from Perkin-Elmer. [3H]RHM-1 (80 Ci/mmol) was synthesized by American Radiolabeled Chemicals, Inc. via O-alkylation of the corresponding phenol precursor ( 18). SW107, N-(9-(6-(5-dimethylamino-1-naphthalensulfonamido))hexyl)-9-azabicyclo[3.3.1]nonan-3α-yl)-N′-(2-methoxy-5-methylphenyl)carbamate, was synthesized as described previously ( 24). Enzyme Free Cell Dissociation Solution was purchased from Chemicon International Inc. Phenylarsine oxide (PAO) was purchased from Sigma Chemical Company. Cell media were purchased from the Washington University Tissue Culture Center. All other chemicals were purchased from Aldrich Chemical Company, Inc., or Sigma Chemical Company.

Chemical synthesis of K05-138, N-9-{6-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl-amino)hexyl}-9-azabicyclo[3.3.1]nonan-3-yl-N′-(2-methoxy-5-methylphenyl)carbamate. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride; 50 mg, 0.25 mmol) was dissolved in 3 mL of acetonitrile and added dropwise to a 3-mL acetonitrile solution of SV119 (100 mg, 0.25 mmol) that was stirred for 1 h at room temperature ( Fig. 1A ). The solvent was removed on a rotavapor, and the residue was purified by preparative TLC (95:5 CH2Cl2/methanol) to give 80 mg of K05-138 (56% yield). Characterization of the structure and purity of K05-138 was determined by nuclear magnetic resonance (NMR) spectroscopy.

Figure 1.
Figure 1.

A, the synthetic scheme for generating K05-138 and SW107. B, excitation and emission spectra of K05-138 in methanol. C, flow-cytometric determination of the internalization of K05-138 in MDA-MB-435 cells with and without blocking by SV119 or (+)-pentazocine. MDA-MB-435 cells were grown in 100-mm dishes for 24 h and then either left untreated or preincubated with 1 to 10,000 nmol/L SV119, or 1 to 10,000 nmol/L (+)-pentazocine, for 1 h at 37°C in a CO2 incubator. After 24 h, 50 nmol/L K05-138 was added, and the cells were reincubated for another 30 min, after which, they were washed, detached from the dishes, and analyzed by flow cytometry. D, quantitative analysis of the internalization of K05-138 with or without SV119 or (+)-pentazocine. The mean fluorescent intensity of the cells treated with either SV119 or (+)-pentazocine was determined from flow cytometry histograms and normalized to a percentage of the mean fluorescent intensity of the cells treated with K05-138 alone.

Receptor binding assays. The sigma-1 and sigma-2 receptor binding affinities of SW107 and K05-138 were determined as previously described ( 25). Briefly, guinea pig brain (sigma-1 assay) or rat liver (sigma-2 assay) membrane homogenates (∼300 μg protein) were diluted with 50 mmol/L Tris-HCl (pH 8.0) and incubated with either ∼5 nmol/L [3H](+)-pentazocine (34.9 Ci/mmol; sigma-1 assay) or 1 nmol/L [3H]RHM-1 (80 Ci/mmol; sigma-2 assay) in a total volume of 150 μL in 96-well plates at 25°C. The concentrations of SW107 and K05-138 ranged from 0.1 nmol/L to 10 μmol/L. After incubating for 60 min, the reactions were terminated by the addition of 150 μL of cold wash buffer [10 mmol/L Tris-HCl, 150 mmol/L NaCl (pH 7.4)] using a 96-channel transfer pipette (Fisher Scientific), and the samples were harvested and filtered rapidly into a 96-well fiberglass filter plate (Millipore) that had been presoaked with 100 μL of 50 mmol/L Tris-HCl at pH 8.0 for 1 h. Each filter was washed thrice with 200 μL of ice-cold wash buffer, and the bound radioactivity was quantified using a Wallac 1450 MicroBeta liquid scintillation counter (Perkin-Elmer). Nonspecific binding was determined in the presence of 10 μmol/L cold haloperidol.

Excitation and emission spectra of K05-138. K05-138 was dissolved in methanol, and the fluorescent excitation and emission spectra were recorded on a Perkin-Elmer LS 50 spectrofluorometer. To determine the excitation spectra, K05-138 was illuminated at wavelengths ranging from 200 to 500 nm, and the fluorescent emission intensity was collected at 530 ± 4 nm. To determine the emission spectra, the excitation wavelengths were set to 260, 340, and 460 nm, and the emission spectra were recorded.

Cell culture conditions. EMT-6 mouse breast tumor cells were cultured in DMEM, supplemented with 10% fetal bovine serum and a 1× penicillin/streptomycin solution. MDA-MB-435 human breast tumor cells were grown in MEM containing 10% fetal bovine serum, 2 mmol/L l-glutamine, 1 mmol/L sodium pyruvate, 1× nonessential amino acids (NEAA), 2% MEM vitamins, and 1× penicillin/streptomycin solution. The cells were maintained at 37°C in a humidified incubator with a 5% CO2/95% air atmosphere. EMT-6 cells or MDA-MB-435 cells were seeded on 35-mm glass-bottom dishes at 2 × 105 cells per dish for 24 h before initiating any treatment.

Two-photon microscopy. A two-photon scanning microscope (Zeiss LSM 510 NLO META) was used. The excitation of wavelength of the Ti/sapphire Chameleon XR laser (Coherent) was set at 720 nm for SW107, and the emission was collected using a 480–520-nm bandpass filter ( 24). MitoTracker, ER-Tracker, and LysoTracker were excited using the 543-nm line from a helium-neon laser, and the emission was collected using a 565–615-nm bandpass filter. FM 1-43FX was excited at 543 nm, and the emission was collected using a 560-nm bandpass filter. The cells were viewed with a 40 × 1.20 numerical aperture (NA) water objective lens. To reduce interchannel cross-talk, a multitracking technique was used. The pixel acquisition time was 1.6 μs. The optical slice thickness was 0.7 μm. Images were taken at a resolution of 1,024 × 1,024 pixels. Two photon scanning parameters were set up so that the cells in the well without the compounds had no fluorescent signal. The cells in the well with SW107 only displayed a green signal, and the cells in the well with mitoTracker, ER-Tracker, or LysoTracker only displayed a red signal. We then used these parameters to scan the cells treated with both compounds.

Confocal microscopy. A confocal laser scanning microscope (Carl Zeiss GmbH Pascal Vario Two UGB) was used. K05-138 was excited using the 488-nm line from an argon laser, and the emission collected with a 505- to 530-nm bandpass filter. MitoTracker, ER-Tracker, or LysoTracker was excited using the 543-nm line from a helium-neon laser, and the emission collected with a 560-nm long-pass filter. The membrane marker FM 4-64FX was excited with a 543-nm line, and the emission was collected with a 560-nm long-pass filter. To reduce interchannel cross-talk, a multitracking technique was used. Image acquisition was done using 40 × 1.20 NA water objective lens. The pixel acquisition time was 1.6 μs. The optical slice thickness was 0.7 μm. Images were taken at a resolution of 1,024 × 1,024 pixels. Confocal scanning parameters were set up so that the cells in the well without the compounds had no fluorescent signal. The cells in the well with K05-138 only displayed a green signal, and the cells in the well with mitoTracker, ER-Tracker, or LysoTracker only displayed a red signal. We then used these parameters to scan the cells treated with both compounds.

Flow cytometry. Flow-cytometric analysis was done using a FACScan (Becton Dickinson) equipped with an air-cooled argon laser using an excitation wavelength of 488 nm and an emission wavelength of 550 nm.

Kinetic study of the internalization of K05-138 in MDA-MB-435 cells. MDA-MB-435 cells were plated in 35-mm-diameter glass-bottom dishes at 2 × 105 cells per dish. After incubating for 24 h, 100 nmol/L K05-138 was added, and images were taken at 25-s intervals. Live cells were monitored using an inverted confocal microscope (Carl Zeiss GmbH Pascal Vario Two UGB) with an excitation wavelength of 488 nm, and the fluorescence was collected with a 505- to 530-nm bandpass filter. Three optical slices at 2-μm intervals were scanned using the Z-stack function of the confocal software. Image acquisition was done using a 40 × 1.20 NA water objective lens. The fluorescent intensity for each cell was calculated as the total intensity of the three optical slices. About 20 cells were analyzed in each dish. The average intensity of the cells versus time was fitted by Eq. A using the PRISM software purchased from GraphPad Software, Inc.:Mathwhere I is the fluorescent intensity at time point T, Imax is the maximum fluorescent intensity, and T1/2 is the time at which the intensity, I, equals one-half of Imax.

Statistical analyses. The results are expressed as the mean ± SD of two or three independent experiments done in triplicate. Differences among groups were statistically analyzed by a one-way ANOVA followed by a Bonferroni's post hoc t test. Comparisons between two experimental groups were done using a two-tailed Student's t test. A P value of <0.05 was considered significant.

Results

Chemical synthesis and characterization of the sigma-2 selective fluorescent ligands. We previously reported the synthesis of the fluorescent ligand, SW107 ( Fig. 1A), which was prepared by reacting dansyl chloride with the corresponding primary amine, SV119 ( 24). SW107 was found to be highly selective for sigma-2 receptors (Kiσ2 = 148 nmol/L) versus sigma-1 receptors (Kiσ1 = 12,600 nmol/L). SW107 had a peak excitation wavelength of 333 nm and an emission spectrum with a range of 480 to 520 nm ( 24). Based on these data, we chose SW107 to specifically image the distribution of sigma-2 receptors in cells using two-photon microscopy.

To prepare a sigma-2 fluorescent probe that can be used with confocal microscopy, SV119 was condensed with the fluorophore NBD chloride to form the sigma-2 selective fluorescent ligand, K05-138 ( Fig. 1A).

In vitro binding studies were then conducted to determine the affinity of K05-138 for the sigma-1 and sigma-2 receptors. The inhibition constant for sigma-2 receptors (Kiσ2) was 45 nmol/L as determined by inhibiting the binding of [3H]RHM-1 to rat liver membrane homogenates. K05-138 also had a low affinity for sigma-1 receptors (Kiσ1 = 1,100 nmol/L). The excitation and emission spectra of K05-138 in methanol were obtained using a spectrofluorometer. K05-138 displayed excitation peaks at three different wavelengths, 260, 340, and 460 nm, respectively ( Fig. 1B). The maximum emission wavelength for all three peak excitation wavelengths was 520 nm ( Fig. 1B). Based on these data, we chose to perform the confocal microscopy studies with K05-138 using an excitation wavelength of 460 nm and an emission wavelength of 520 nm.

To study whether K05-138 binds to sigma-2 receptors and not sigma-1 receptors in tumor cells, a series of blocking experiments were done with sigma-1 and sigma-2 selective ligands. MDA-MB-435 human breast tumor cells were preincubated with SV119, a sigma-2 selective ligand, or (+)-pentazocine, a sigma-1 selective ligand, for 1 h at 37 °C at various concentrations ranging from 1 to 10,000 nmol/L. The cells were then treated with 50 nmol/L of K05-138 for 30 min, and the fluorescent intensity of the labeled cells was analyzed by flow cytometry. The data indicate that SV119 blocked the binding of K05-138 in a concentration-dependent manner ( Fig. 1C and D). Approximately 80% of the binding of K05-138 was blocked by SV119 at a concentration of 10 μmol/L. In contrast, (+)-pentazocine did not block the binding of K05-138 at 10 μmol/L, the highest concentration used in this study. These flow cytometry results were confirmed by confocal microscopy (data not shown). Taken together, the data show that K05-138 selectively binds to the sigma-2 receptors in tumor cells.

Colocalization of SW107 and subcellular organelle markers by two-photon microscopy. The subcellular localization of sigma-2 receptors in both EMT-6 mouse breast cancer cells and MDA-MB-435 human breast cancer cells was studied with two-photon microscopy using SW107 and fluorescent markers of several subcellular organelles. EMT-6 or MDA-MB-435 cells were incubated with 200 nmol/L SW107 and one of three subcellular organelle markers using the concentrations recommended by the manufacturer: the mitochondria marker, MitoTracker Red CMXRos (50 nmol/L), the endoplasmic reticulum marker, ER-Tracker Red (500 nmol/L), or the lysosome marker, LysoTracker Red DND-99 (75 nmol/L). After incubating at 37°C for 2 h, live cells were imaged by two-photon microscopy. Our results show that SW107 is distributed throughout the cytoplasm of the cells, but not in the nucleus ( Figs. 2 and 3 ). The SW107 staining is highly punctated, suggesting that the label has been sequestered in small membrane-bound compartments. SW107 colocalizes with the MitoTracker, ER-Tracker, and LysoTracker in both EMT-6 ( Fig. 2A–C) and MDA-MB-435 cells ( Fig. 3A–C). These data show that sigma-2 receptors are localized in the mitochondria, lysosomes, and endoplasmic reticulum.

Figure 2.
Figure 2.

Determination of the intracellular distribution of SW107 in EMT-6 cells with and without MitoTracker (A), LysoTracker (B), or ER-Tracker (C) using two-photon microscopy. EMT-6 cells were incubated with 200 nmol/L SW107 and either 50 nmol/L MitoTracker, 500 nmol/L ER-Tracker, or 75 nmol/L LysoTracker. After incubating for 2 h at 37°C, live cells were imaged by two-photon microscopy.

Figure 3.
Figure 3.

Determination of the intracellular distribution of SW107 in MDA-MB-435 cells with and without MitoTracker (A), LysoTracker (B), ER-Tracker (C), or the membrane tracker, FM 1-43FX (D) using two-photon microscopy. MDA-MB-435 cells were incubated with 200 nmol/L SW107 and either 50 nmol/L MitoTracker, 500 nmol/L ER-Tracker, or 75 nmol/L LysoTracker for 2 h at 37°C. MDA-MB-435 cells were also incubated with 200 nmol/L SW107 and 5 μg/mL of the membrane tracker, FM 1-43FX, for 5 min at 0°C. After the incubation period, live cells were imaged by two-photon microscopy.

MDA-MB-435 cells were also incubated for 5 min at 0°C with 200 nmol/L of SW107 in HBSS buffer, which does not contain Ca2+ and Mg2+, either with or without 5 μg/mL of the plasma membrane marker, FM 1-43FX. The two-photon microscopy results show that SW107 colocalizes with the plasma membrane marker ( Fig. 3D), indicating that sigma-2 receptors also exist in the plasma membrane.

Colocalization of K05-138 and subcellular organelle markers by confocal microscopy. The subcellular localization of K05-138 in both EMT-6 mouse breast cancer cells and MDA-MB-435 human breast cancer cells was studied using confocal microscopy. EMT-6 or MDA-MB-435 cells were incubated with 100 nmol/L K05-138 and each of the four subcellular organelle markers as described above for the two-photon microscopy experiments. The results show that K05-138 colocalizes with the fluorescent markers of the mitochondria, lysosomes, endoplasmic reticulum, and plasma membrane in EMT-6 ( Fig. 4A–C ) and MDA-MB-435 ( Fig. 5A–D ) cells. These results are consistent with the data from the two-photon microscopy experiments.

Figure 4.
Figure 4.

Determination of the intracellular distribution of K05-138 in EMT-6 cells with and without MitoTracker (A), LysoTracker (B), or ER-Tracker (C) using confocal microscopy. EMT6 cells were incubated with 100 nmol/L K05-138 and either 50 nmol/L MitoTracker, 500 nmol/L ER-Tracker, or 75 nmol/L LysoTracker. After incubating for 2 h at 37°C, live cells were imaged by confocal microscopy.

Figure 5.
Figure 5.

Determination of the intracellular distribution of K05-138 in MDA-MB-435 cells with and without MitoTracker (A), LysoTracker (B), ER-Tracker (C), or the membrane tracker, FM 4-64FX (D) using confocal microscopy. MDA-MB-435 cells were incubated with 100 nmol/L K05-138 and either 50 nmol/L MitoTracker, 500 nmol/L ER-Tracker, or 75 nmol/L LysoTracker for 2 h at 37°C. MDA-MB-435 cells were also incubated with 100 nmol/L K05-138 and 5 μg/mL of the membrane tracker, FM 4-64FX, for 5 min at 0°C. After the incubation period, live cells were imaged by confocal microscopy.

Kinetic studies of the internalization of K05-138. The time course for internalization of K05-138 in MDA-MB-435 cells was monitored by confocal microscopy ( Fig. 6 ). Figure 6A shows some of the time-lapsed confocal images acquired at 25-s intervals. The fluorescent intensity of K05-138 in the cells increased rapidly over the first 2 min of exposure and reached a plateau after 5 min. After fitting the data, the average half time for internalization of the receptor (T1/2) was 16 s ( Fig. 6B).

Figure 6.
Figure 6.

Elucidation of the mechanism and kinetics for the internalization of K05-138 in MDA-MB-435 cells. A, real-time imaging of K05-138 internalization. MDA-MB-435 cells were plated in 35-mm glass-bottom dishes at 2 × 105 cells per dish. After incubating for 24 h, 100 nmol/L K05-138 was added to each dish, and the images were taken at 25-s intervals. B, fluorescent intensity as a function of internalization time. The experimental data (○) were fitted by Eq. A (see Materials and Methods) to obtain the solid line from which the value of T1/2 (16 s) was calculated. The data are the average of three independent experiments. C, flow-cytometric determination of the inhibition of K05-138 internalization by PAO. MDA-MB-435 cells were preincubated with or without 5 or 10 μmol/L PAO for 30 min at 37°C and then incubated with 50 nmol/L K05-138 for an additional 5 min. After washing, the cells were detached from the dishes and analyzed by flow cytometry. The internalization of K05-138 was significantly reduced by both 5 and 10 μmol/L PAO. *, P < 0.005.

To study whether the internalization of sigma-2 receptors is mediated by endocytosis, we examined the effect of PAO, a well-characterized endocytosis inhibitor ( 26), on the internalization of K05-138. MDA-MB435 cells were pretreated with 5 or 10 μmol/L PAO for 30 min and then treated with 50 nmol/L K05-138 in the absence or presence of PAO for an additional 5 min. Flow-cytometric analysis ( Fig. 6C) showed that 5 and 10 μmol/L PAO significantly (P < 0.005) blocked internalization of K05-138 by 31% and 38%, respectively. These data show that ∼40% of the sigma-2 receptors were internalized by an endocytosis mechanism, whereas the remaining ∼60% was internalized by other mechanisms such as passive diffusion.

Discussion

In the current study, we describe the development and use of two fluorescent sigma-2 ligands, SW107 and K05-138, to image the subcellular localization of sigma-2 receptors in live cells. Colocalization studies of the fluorescent sigma-2 receptor ligands with fluorescent markers of the mitochondria, lysosomes, endoplasmic reticulum, and plasma membrane were conducted using two-photon and confocal microscopy. The goal of these studies was to provide information on the subcellular localization of the sigma-2 receptor and to compare our results with previous studies that examined the role of sigma-2 receptor ligands in producing cell death.

The first step in this process involved the pharmacologic characterization of the fluorescent compounds with respect to their affinity and selectivity for sigma-2 receptors. The results of our receptor binding studies showed that the SW107 inhibition constants (Ki) are 148 nmol/L for the sigma-2 receptors and 12,600 nmol/L for the sigma-1 receptors ( 24). The K05-138 Ki values are 45 nmol/L for the sigma-2 receptors and 1,100 nmol/L for the sigma-1 receptors. Based on these Ki values, SW107 at a final concentration of 200 nmol/L and K05-138 at a final concentration of 100 nmol/L were used in the microscopic imaging studies. The fluorescent ligands at the chosen concentrations should selectively bind to sigma-2 receptors. This was shown by blocking experiments that showed that the sigma-2 selective ligand, SV119, blocked K05-138 internalization into cells, whereas the sigma-1 selective ligand, (+)-pentazocine, did not ( Fig. 2C and D). These results indicate that our fluorescent compounds are useful probes for specifically imaging sigma-2 receptors in cells.

The data presented here also show that sigma-2 ligands colocalize with an endoplasmic reticulum marker, indicating that sigma-2 receptors reside in the endoplasmic reticulum ( Figs. 2– 5). The endoplasmic reticulum serves as a dynamic Ca2+ storage pool ( 27). Ca2+ is an important intracellular signal for cellular processes such as growth, differentiation, and apoptosis ( 28). Sigma-2 ligands have been shown to stimulate rapid and transient Ca2+ release from a thapsigargin-sensitive store in the endoplasmic reticulum ( 29). The time course of K05-138 entry into cells ( Fig. 6A and B) is comparable to that of Ca2+ release from the endoplasmic reticulum reported previously ( 29). Also, the Ca2+ release channels (InsP3 and ryanodine receptors) and the sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) pumps reside in the endoplasmic reticulum membrane and regulate Ca2+ release ( 27, 30). Additional research will be required to determine if sigma-2 receptors interact with IP3 receptors, ryanodine receptors or SERCAs, directly or indirectly, to regulate Ca2+ release from the endoplasmic reticulum.

The data presented here also show that the sigma-2 ligands colocalize with the LysoTracker, indicating that sigma-2 receptors are localized in lysosomes ( Figs. 2– 5). In a recent report ( 23), the sigma-2 selective ligand, siramesine, has been shown to cause lysosomal leakage and effectively induce caspase-independent programmed cell death in tumor cells. An inhibitor of the lysosome protease, cathepsin B, partially blocked siramesine-induced cell death. The lysosomal localization of sigma-2 receptors in the present study is consistent with a lysosomal role in sigma-2 ligand-induced cell death. During recent years, there has been growing evidence to suggest that lysosomal proteases, such as cathepsins, calpains, and granzymes, contribute to apoptosis ( 31). Under physiologic conditions, these proteases are found within the lysosomes and are released into the cytoplasm after exposure to cell-damaging agents, thereby triggering a cascade of intracellular degradative events. The localization of our fluorescent sigma-2 receptor probes in the lysosomes is consistent with the hypothesis that the sigma-2 receptor ligands induce cell death partially by targeting lysosomes to cause lysosome damage, the release of proteases, and eventually, cell death ( 23). Nevertheless, we cannot eliminate the possibility that the presence of the fluorescent sigma-2 probes in the lysosomes might reflect lysosomal degradation of sigma-2 receptors ( 32).

The confocal and two-photon microscopy studies reported here also show that sigma-2 ligands colocalize with the mitochondria marker, MitoTracker Red ( Figs. 2– 5). These data suggest that sigma-2 receptors also reside in the mitochondria. These results are consistent with a previous report showing that purified mitochondria contain a high density of sigma-2 receptors ( 33). In addition, the results from several laboratories, including ours, have shown that sigma-2 receptor ligands induce apoptosis through caspase-dependent pathways in several tumor cell lines ( 21– 23, 34). It has been well established that the release of cytochrome c from the mitochondria is a key step in the intrinsic apoptotic pathway ( 35). In fact, the loss of mitochondrial membrane potential and induction of caspase-dependent apoptosis by sigma-2 selective ligands has been reported in SK-N-SH neuroblastoma cells ( 22). Taken together, these data suggest that sigma-2 ligands may induce, either directly or indirectly, the intrinsic apoptotic pathway by interacting with mitochondrial sigma-2 receptors.

The data presented here also show that SW107 and K05-138 colocalize with plasma membrane markers, indicating that sigma-2 receptors reside in the plasma membrane ( Figs. 3 and 5). This result is consistent with the previous reports that sigma-2 receptors are enriched in the membrane preparations of cells and tissues ( 11, 36). Our data are also consistent with previous studies suggesting that sigma-2 receptors exist in lipid rafts ( 36), which are largely found in the plasma membrane ( 37). Lipid rafts are organized microdomains in cell membranes and are enriched with cholesterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins ( 37– 39). Lipid rafts play an important role in the signaling associated with a variety of cellular events such as adhesion, motility, and membrane trafficking ( 37, 40). Thus, our fluorescent probes may prove to be useful tools for studying sigma-2 receptors in lipid rafts using two-photon and confocal microscopy.

An interesting and unexpected observation in the current study was the rapid internalization of the sigma-2 fluorescent probes, suggesting that sigma-2 receptors are internalized via endocytosis ( Fig. 6A and B). The rapid internalization rate of K05-138 (T1/2 = 16 s) is comparable with that of other molecules that undergo receptor-mediated endocytosis ( 41, 42). However, because K05-138 is a lipophilic small molecule, it also crosses the plasma membrane via passive diffusion because PAO only reduced the uptake of this fluorescent probe by ∼40% ( Fig. 6C). The apparent rapid internalization of sigma-2 receptors via endocytosis suggests that sigma-2 selective ligands may potentially serve as receptor-mediated probes for delivering cytotoxic agents to solid tumors.

In conclusion, SW107 and K05-138 are fluorescent probes that can be used to study the localization and function of sigma-2 receptors. Our data indicate that sigma-2 receptors are localized in the mitochondria, lysosomes, endoplasmic reticulum, and plasma membrane. After ligand binding, sigma-2 receptors residing on plasma membranes seem to be rapidly internalized into cells by an endocytotic pathway. Thus, the data presented here suggest that sigma-2 selective ligands may not only be used to image solid tumors and determine their proliferative status with PET and single-photon emission computed tomography, but they also may have the potential to serve as cancer chemotherapeutic agents.

Acknowledgments

Grant support: Funded in part by CA 102869.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Mark P. Goldberg, Krzysztof Hyrc, and Samuel Achilefu for assistance with confocal and two-photon microscopy.

Footnotes

    • Received October 16, 2006.
    • Revision received March 29, 2007.
    • Accepted May 11, 2007.

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    Cancer Research: 67 (14)
    July 2007
    Volume 67, Issue 14

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    Subcellular Localization of Sigma-2 Receptors in Breast Cancer Cells Using Two-Photon and Confocal Microscopy
    Chenbo Zeng, Suwanna Vangveravong, Jinbin Xu, Katherine C. Chang, Richard S. Hotchkiss, Kenneth T. Wheeler, Duanwen Shen, Zhi-Ping Zhuang, Hank F. Kung and Robert H. Mach
    Cancer Res July 15 2007 (67) (14) 6708-6716; DOI: 10.1158/0008-5472.CAN-06-3803
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